The present invention, in some embodiments thereof, relates to therapy and, more particularly, but not exclusively, to methods and compositions for the treatment of Hepatitis C Virus (HCV) related diseases such as chronic HCV infection.
HCV is a positive-stranded RNA virus which has been classified as a separate genus in the Flaviviridae family. All members of the Flaviviridae family have enveloped virions that contain a positive stranded RNA genome encoding all known virus-specific proteins via translation of a single, uninterrupted, open reading frame. The single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3,000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins. In the case of HCV, the generation of mature non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one cleaves at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a co-factor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B is a RNA-dependent RNA polymerase that is involved in the replication of HCV.
Infection by hepatitis C virus (HCV) is a compelling human medical problem. HCV is recognized as the causative agent for most cases of non-A, non-B hepatitis, with an estimated human sero-prevalence of 3% globally. Nearly four million individuals may be infected in the United States alone. Upon first exposure to HCV only about 20% of infected individuals develop acute clinical hepatitis while others appear to resolve to the infection spontaneously. In almost 70% of instances, however, the virus establishes a chronic infection that persists for decades. This usually results in recurrent and progressively worsening liver inflammation, which often leads to more severe disease states such as cirrhosis and hepatocellular carcinoma.
The combination of a pegylated interferon (e.g., peg-IFN alpha-2a/b) and twice-daily oral doses of ribavirin, an anti-viral agent, is the current standard of care for the treatment of chronic HCV infection. Patients who will ultimately achieve a sustained virologic response to peg-IFN and ribavirin therapy usually develop a rapid decline in HCV-RNA levels after initiation of therapy, with levels becoming undetectable within 4-24 weeks. Liver enzyme levels become normal, and histologic findings improve markedly. With the above-mentioned combination therapy, approximately 75% to 80% of patients with HCV genotype 2 or 3 infection and 40% to 50% of those with genotype 1 infection achieve a sustained virologic response (SVR) [Sherman K. E., Clinical Need and Therapeutic Targets for New HCV Agents, in The Future of HCV: Small molecules in Development for Chronic Hepatitis C, Clinical Care Options LLC, 2007].
However, success rate of this combined therapy is limited as its outcome is highly dependent on the infecting HCV genotype. This treatment is effective in fewer than 50% of patients infected with HCV genotype 1 or 4, the most represented genotypes in Europe and USA. In many cases, non-response is related to host or viral factors that impair activation of the host's innate, interferon-driven immune response.
Others may achieve viral reduction during therapy but cannot tolerate full therapeutic doses or an adequate duration of treatment because of cytopenia, fatigue, or other adverse effects of treatment. Indeed, dose modifications for these reasons are required in 35% to 42% of treated patients, and approximately one third of these patients eventually discontinue treatment altogether. These dose reductions, temporary interruptions, and aborted treatment courses reduce the chance of achieving SVR.
Finally, the combination of peg-IFN and ribavirin is contraindicated altogether in many patients who are in need of anti-HCV therapy. Contraindications for therapy include severe cytopenia, hepatic decompensation, renal insufficiency, poorly controlled autoimmune disease, severe cardiopulmonary disease, and active psychological problems. [Davis G. L., Investigational Small-Molecule Agents for the Treatment of to Chronic Hepatitis C, in The Future of HCV: Small molecules in Development for Chronic Hepatitis C, Clinical Care Options LLC, 2007].
Briolant et al. [Antiviral Research 61 (2004) 111-117 teach that a combination of IFN-α2b and ribavirin has a subsynergistic anti-viral effect on CHIKV and SFV.
Alternative therapies for the treatment of HCV related diseases have been developed. U.S. Pat. No. 6,849,254 discloses a combination therapy including the administration of interferon alfa and ribavirin for a time sufficient to lower HCV-RNA, in association with an antioxidant for a time sufficient to improve ribavirin-related hemolysis.
U.S. Pat. No. 7,115,578 discloses a combination therapy comprising administering a therapeutically effective amount of ribavirin derivatives and a therapeutically effective amount of interferon-alfa. U.S. Pat. No. 7,410,979 discloses a synergistically effective combination therapy of dihaloacetamide compounds and interferon or ribavirin against HCV infection. U.S. Pat. No. 7,671,017 discloses the use of cyclosporine and pegylated interferon for treating HCV.
Chloroquine is a well known lysosomotropic agent, currently attracting many hopes in terms of antiviral therapy as well as in antitumoral effect because of its pH-dependent inhibiting action on the degradation of cargo delivered to the lysosome, thus effectively disabling this final step of the autophagy pathway.
Hydroxychloroquine (HCQ) is a chemical derivative of chloroquine (CQ) which features a hydroxyethyl group instead of an ethyl group.

HCQ has been classified as an effective anti-malarial medication, and has shown efficacy in treating systemic lupus erythematosus as well as rheumatoid arthritis and Sjögren's Syndrome. While HCQ has been known for some time to increase lysosomal pH in antigen presenting cells, its mechanism of action in inflammatory conditions has been only recently elucidated and involves blocking the activation of toll-like receptors to on plasmacytoid dendritic cells (PDCs).
A direct comparison of the therapeutic effect of CQ and HCQ is quite difficult but it has been suggested that hydroxychloroquine was one-half to two-thirds as effective as chloroquine in treating rheumatologic diseases and one-half as toxic [Scherbel A L et al., Cleve Clin Q, 1958, 25:95]. Since chloroquine appears to be much more retinotoxic frequent use of hydroxychloroquine is increasing [Rynes R. I., British Journal of Rheumatology, 1997; 36:799-805].
Chandramohan M. et al. [Indian J Pharm Sci 2006; 68:538-40] have reported the screening of chloroquine and hydroxychloroquine for potential in vitro antiviral activity against HCV in Huh 5-2 cells, and showed that chloroquine was able to reduce the viral RNA to below 7% and promoted cell growth to more than 91% with respect to the untreated control at the concentration of 10.75 μM, and that hydroxychloroquine was able to reduce the viral RNA to below 7% and promoted cell growth to more than 81% with respect to the untreated control at the concentration of 6.6 μM. Chandramohan M. et al. neither demonstrate that HCQ may be used in combination with an antiviral agent to treat HCV related diseases, nor characterize the effect of such a combination (antagonist, additive, or synergistic).
Freiberg et al. [Journal of General Virology (2010), 91, 765-772] have evaluated the antiviral efficacy of chloroquine, individually and in combination with ribavirin, in the treatment of NiV and HeV infection in in vivo experiments, using a golden hamster model, and have reported that while both drugs exhibit a strong antiviral activity in inhibiting viral spread in vitro, they did not prove to be protective in the in vivo model. Ribavirin delayed death from viral disease in NiV-infected hamsters by approximately 5 days, but no significant effect in HeV-infected hamsters was observed. Chloroquine did not protect hamsters when administered either individually or in combination with ribavirin, the latter indicating the lack of a favorable drug-drug interaction.
Zuckerman et al. [BioDrugs 2001; 15(9), pp. 574-584] suggest that oral administration of drugs such as HCQ, corticosteroids and other anti-inflammatory agents can be combined with anti-viral therapy for controlling HCV-related arthritis.
Mizui et al. [J Gastroenterol. 2010 February; 45(2):195-203. Epub 2009 Sep. 17] have later reported that treatment of cells transfected with HCV replicon with chloroquine suppressed the replication of the HCV replicon in a dose-dependent manner. It was shown that a treatment with chloroquine, a known inhibitor of autophagy, and interferon-alfa enhanced the antiviral effect of the interferon, thereby preventing re-propagation of HCV replicon. Mizui et al. did not demonstrate any synergistic or additive effect of the combination of CQ/interferon alfa, and did not relate to HCQ as a putative drug for HCV combination therapy.
A recent approach of gene expression profiling of JFH1 HCV infected Huh7 cells showed that infection clearly modulates expression of host genes involved in several cellular processes such as ER stress response, apoptosis, p53 signaling, detoxification, intracellular lipid metabolism, protein synthesis and degradation, post translational processes or cytoskeleton organization.
The link between autophagy, a mechanism for cell survival in response to cellular stress role in cell death, and viral replication, including in cases of HCV infection, is currently being investigated.
Autophagy, a cellular pathway leading to components self-degradation, is known to be activated in response to stress, including ER stress initiated after viral infection. Although autophagy provides protection against various infections, and has been described as a component of the innate immune response, several bacteria and viruses, including HCV, have developed strategies to subvert autophagic processes to facilitate their own replication [Schmid & Munz, Immunity 2007, 27:11-21; Wileman, Science 2006, 312:875-878]. It was found that silencing autophagy-related genes significantly blunts the replication of HCV [Mizui et al., 2010 supra] and decreases the production of infectious HCV particles. It has been suggested that induction of autophagy by HCV impairs the innate immune response, and disruption of autophagy in HCV-infected hepatocytes activates the interferon signaling pathway and enhances the innate immune response [Shrivastava et al., Hepatology 2011, 53:406-414].
HCV infection, in vitro and in vivo, induces ER stress and triggers autophagy through the induction of unfolded protein response (UPR) including the downstream IRE1, ATF6, and EIF2AK3/PERK signaling pathways [Sir et al., Hepatology 2008, 48:1054-1061]. However, HCV-induced autophagic process was thought to be incomplete since it does not lead to protein degradation [Sir et al., Hepatology 2008, 48:1054-1061].
Ke P Y. et al. have reported that a completed autophagic process totally suppresses innate antiviral immunity, through a blockade of the endogenous IFN response, allowing HCV RNA replication.
Although the main autophagy proteins seem to be proviral factors required for the translation of incoming HCV RNA, and thereby for the initiation of HCV replication, it has been suggested that autophagy is not required once infection is established [see, for example, Dreux et al., PNAS 2009, 106:14046-14051].
However, neither the host molecular mechanisms involved in response to HCV infection, nor the molecular basis of the antiviral activity of CQ and its interplay with autophagy have been clearly elucidated heretofore.
Recently, it has been suggested that autophagy is involved also in cancer therapy, as being induced by chemotherapy such as DNA-damaging chemotherapy, radiation therapy, and molecularly targeted therapies, and that chloroquine derivatives such as HCQ may be used to prevent the chemotherapy-induced autophagy [Ravi K. Amaravadi, J Clin Invest. 2008; 118(12):3837-3840].
Additional background art includes Jackson et al. [PLoS Biol 2005, 3:e156], Wong et al. [J Virol 2008, 82:9143-9153], Khakpoor et al. [J Gen Virol 2009, 90:1093-1103], Lee et al. [Virology 2008, 378:240-248], Prentice et al. [J Bio Chem 2004, 279:10136-10141], and Kroemer et al. [Nat Rev Mol Cell Biol 2008, 9:1004-1010].